Abstract

PURPOSE:

Cancers overexpressing the HER2/neu gene are usually more aggressive and are associated with poor prognosis. Although trastuzumab has significantly improved the outcome, many tumors do not respond or acquire resistance to current therapies. To provide an alternative HER2-targeted therapy, we have developed and characterized a novel recombinant protein combining an HER2-specific Affibody and modified Pseudomonas aeruginosa exotoxin A (PE 38), which, after binding to HER2, is internalized and delivered to the cytosol of the tumor cell, where it blocks protein synthesis by ADP ribosylation of eEF-2.

EXPERIMENTAL DESIGN:

The effect of the Affitoxin on cell viability was assessed using CellTiter-Glo (Promega). To assess HER2-specific efficacy, athymic nude mice bearing BT-474 breast cancer, SK-OV-3 ovarian cancer, and NCI-N87 gastric carcinoma xenografts were treated with the Affitoxin (HER2- or Tag-specific), which was injected every third day. Affitoxin immunogenicity in female BALB/c mice was investigated using standard antibody production and splenocyte proliferation assays.

RESULTS:

In vitro experiments proved that HER2-Affitoxin is a potent agent that eliminates HER2-overexpressing cells at low picomolar concentrations. Therapeutic efficacy studies showed complete eradication of relatively large BT-474 tumors and significant effects on SK-OV-3 and NCI-N87 tumors. HER2-Affitoxin cleared quickly from circulation (T(1/2) < 10 minutes) and was well tolerated by mice at doses of 0.5 mg/kg and below. Immunogenicity studies indicated that HER2-Affitoxin induced antibody development after the third injected dose.

CONCLUSIONS:

Our findings showed that HER2-Affitoxin is an effective anticancer agent and a potential candidate for clinical studies.

Treating disseminated ovarian SK-OV-3 tumors with HER2-Affitoxin.Mice bearing peritoneal SK-OV-3-luc-D3 tumors were treated with vehicle (control) or received 6 doses of HER2-Affitoxin (0.25 mg/kg) every third day, as indicated by arrows. A, BLI images taken 11 days after the last dose of HER2-Affitoxin was injected. B, Changes in mean bioluminescence intensity during the course of the experiment (arrows indicate HER2-Affitoxin injection time points and asterisks represent the statistical significance between groups; p < 0.05, as determined by t-student test). C, Average and signal distributions in control- and HER2-Affitoxin-treated groups 11 days after the last dose was injected (p value was determined by one-tailed t-student test). Control and treated groups of mice were injected IP with D-Luciferin solution (75 mg/kg). Images were acquired using IVIS Lumina imaging systems 8–10 minutes after D-Luciferin administration.

Induction of HER2-Affitoxin antibodies.Mice were injected with 0.25 mg/kg of HER2-Affitoxin, or vehicle alone (control) every 2 weeks for a total of 6 weeks. Plasma samples were collected 10 days after each injection and assayed with ELISA for the presence of HER2-Affitoxin antibodies. Plasma from control mice was subjected to HER2-Affitoxin (HER2-Affitoxin control). Data are depicted as the average fold increase ± standard error of the mean (SEM) of anti-toxin antibodies measured from mice receiving toxin compared to control mice (n = 3–4 mice per group.)